The overall goal of this proposal is to understand mechanisms of cell-mediated immunity, including recognition of infected host cells and the killing of microbial pathogens. This proposal represents a collaboration between Dr. Barry Bloom of Albert Einstein College of Medicine, New York, and Dr. Robert Modlin of UCLA, two investigators who have long worked closely and productively, that addresses national research priority areas identified by NIH. Previous work from several laboratories has demonstrated that lymphokine responses and macrophage activation are necessary, but not sufficient, for resistance to many intracellular pathogens. Preliminary data from these investigators indicate that CD8+ cytotoxic T lymphocytes (CTL) have the capability not only to kill infected cells, but also induce mechanisms enabling them directly to reduce the viability of a variety of bacterial pathogens. This suggests that one or more molecules within cytotoxic granules of CTL have antimicrobial activity. One focus of this proposal will be on a newly identified molecule of CTL granules, granulysin, which preliminary data indicate is microbicidal for several gram-positive and gram-negative bacterial pathogens, including M. tuberculosis. One aim is to determine the mechanism by which granulysin kills such pathogens, to understand how this molecule is delivered to infected cells, and how it contributes to protection against intracellular pathogens. A second aim is to elucidate how a pathogen that resides primarily within a vacuolar compartment of host macrophages can present antigens through the MHC class I pathway to CTL. Finally, the applicants propose to elucidate the specific role of MHC class I-restricted CTL in protection against intracellular pathogens, studying particularly M. tuberculosis models, including transgenic mice unable to express MHC class I or CD1. From these studies, they propose to develop a surrogate endpoint for MHC class I-restricted CTL that could be used to monitor the ability of candidate vaccines to engender CTL and protective immunity in humans. The overall goal is to contribute to an understanding of how cell- mediated immune responses to intracellular pathogens engender resistance that will allow development of new approaches to immunization.